The periodontal pathogen, Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, expresses several complex multi-gene cytotoxin systems that negatively impact specific types of cells important to the health of the human periodontium. These systems include genetic loci for a leukotoxin and a cytolethal distending toxin (Cdt). This impressive repertoire of virulence factors has not been found in other oral bacterial pathogens. Our prior studies demonstrated that strains of A. actinomycetemcomitans, isolated from subjects that have converted from a healthy to diseased periodontal state, produce a Cdt that causes DNA damage and arrest of cell cycle progression of primary human gingival epithelial cells (HGEC). This Cdt is an atypical AB type toxin composed of three heterologous gene products. The objectives of this application are to understand the structure/function relationships of the Cdt subunits and to define the specific interactions of the subunits with primary differentiated and undifferentiated HGEC. The general hypothesis of our study is that functional domains unique to each subunit contribute to the exquisite sensitivity and target specificity of HGEC. The primary role of CdtA is to recognize and bind to the cell surface receptor for the toxin while that of CdtC is to stabilize CdtB binding in the heterotrimer complex and to facilitate the intracellular transport of this subunit. In the context of colonization and pathogenesis the protective epithelial cell layer that lines the gingiva is the principal target for the Cdt. The epithelial layer is a physical barrier to infection and functions as part of a signaling network that alerts inflammatory cells to microbial assault. The Cdt preferentially inhibits the rapidly proliferating undifferentiated versus differentiated gingival epithelial cells affecting the growth and turnover of this layer. Damage to this layer would allow A. actinomycetemcomitans as well as other opportunistic bacterial species, and/or their products, to more easily reach and attack the underlying connective tissue and infiltrating inflammatory cells. The specific aims of the study are: (1) to identify and characterize specific domains in the CdtA and CdtC subunits required for heterotoxin assembly and binding to susceptible host cells, (2) to assess the contributions of the CdtC subunit in the assembly, intracellular transport and cytotoxicity of CdtB, (3) to use the cdt subunit mutants and Chinese hamster ovary (CHO) cell mutants to obtain detailed information about specific subunit functions and interactions in vivo and (4) to develop and begin to characterize a primary human gingival epithelial cell (HGEC) model to assess the effects of the Cdt. We expect that this approach will advance our ongoing structure/function studies of the Cdt and provide insight into how the Cdt may contribute to the complex cascade of events that lead to the development of periodontal lesions as part of a polymicrobial etiology. This information can be exploited therapeutically to block Cdt activity to reduce the severity of the tissue destruction that is a hallmark of periodontal disease. PUBLIC HEALTH RELEVANCE: The oral bacterial pathogen Aggregatibacter actinomycetemcomitans produces a cytotoxin, known as the cytolethal distending toxin. This toxin causes DNA damage that signals the inhibition of growth of human gingival epithelial cells. Disruption of the protective epithelial cell layer formed in the gingival tissues gives opportunistic bacteria and their products access to the underlying connective tissue and infiltrating inflammatory cells important for the maintenance of a healthy periodontium.